The present invention relates to the establishment of a cultivation condition that is suitable for the large-scale production of pharmacologically active filtrates from a culture of A. camphorata, in particular, by optimizing the agitation rate and/or pH value during the cultivation. The present invention also relates to a process for obtaining pharmacologically active compositions from a culture of A. camphorata through a series of fractionation. This invention is further directed to the uses of the above compositions in the preparation of pharmaceutical compositions.
A. camphorata [(Zang and Su) S.-H. Wu, Ryvarden and T. T. Chang], also known as xe2x80x9cniu-chang-chihxe2x80x9d or xe2x80x9cniu-chang-kuxe2x80x9d in Taiwan, was recently reported as a new fungus species characterized by the cylindrical shape of its basidiospores appearing in fruit bodies, weakly amyloid skeletal hypha, bitter taste and light cinnamon resupinate to pileate basidiocarps, as well as chlamydospores and arthrocnidia in pure culture. The detail characterization and taxonomic position of A. camphorata were described in Chang, T. T. et al., A. cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan, Mycol. Res. 99(6): 756-758 (1995) and Wu, S. -H., et al., A. cinnamomea (xe2x80x9cniu-chang-chihxe2x80x9d), New combination of a medicinal fungus in Taiwan, Bot. Bull. Acad. Sin. 38: 273-275 (1997), the entire disclosure of which is incorporated herein by reference.
In Taiwan folk medicine, A. camphorata is believed to have certain medical effects on conditions caused by poisoning, diarrhea, abdominal pain, hypertension, skin itches and liver cancer. However, because of the stringent host specificity and rarity in nature, as well as the failure of artificial cultivation, xe2x80x9cniu-chang-chinxe2x80x9d is very expensive in local market. Undoubtedly, there exists a great industrial value in developing a cost-effective process for artificial cultivation of this fungus on a large scale.
Recently, the inventors have found that A. camphorata, when subjected to submerged fermentation, exhibits desired pharmacological activity, in particular an anti-tumor activity. As disclosed in U.S. Ser No. 09/566,834, A. camphorata were successfully cultivated on a small scale in a natural medium, namely potato dextrose broth (PDB), and a synthetic medium containing fructose as the major carbon source. The resultant cultures expressed a color appearance of deep dark red with a redness index axe2x89xa73 as measured using Hunter""s coordinate system, which has been shown to coincide with a significant inhibitory effect on the growth of certain tumor cell lines. More importantly, the active substances functioning to the tumor cells, though not identified, were found secreted from the fungal mycelium into the liquid phase of the culture, so that a pharmacologically active composition can be easily harvested from the culture for industrial use.
Accordingly, it would be desirable if such an advantageous process is optimized for large-scale production of the fungal culture. More preferably, the crude filtrate is further fractionated over a desired pharmacological activity so as to obtain useful compositions enriched with the desired activity.
An extensive investigation has been conducted to fulfill the industrial requirements as indicated above. It has now been unexpectedly found that an optimal condition for the industrial-scale cultivation of A. camphorata can be accomplished by carefully setting certain parameters within particular ranges. In this invention, pH value and agitation speed are found to be critical during cultivation.
Accordingly, the first aspect of the present invention is to provide a process for producing an Antrodia camphorata culture having pharmacological activity, comprising:
(a) inoculating a mycelial inocutum of an isolate of Antrodia camphorata into a medium suitable for growth of said isolate to result in a first culture;
(b) subjecting the first culture cultivated from step (a) to a first stage of agitation which is set at a first predetermined rate and for a first period of time to allow further growth of the inoculated isolate, so as to obtain a second culture proliferation with mycelium; and
(c) subjecting the second culture obtained from step (b) to a second stage of agitation which is set at a second predetermined rate different from the first predetermined rate, so as to subject the isolate under physiological stress.
According to the second aspect of the present invention, it is to provide a process for producing an Antrodia camphorata culture having pharmacological activity, comprising:
(a) inoculating a mycelial inoculum of an isolate of Antrodia camphorata into a medium suitable for growth of said isolate; and
(b) cultivating the culture resulting from step (a) by adjusting the pH value of the culture to a range from 4.5 to 5.4 throughout step (b).
Preferably, the pH value of the A. camphorata culture is adjusted within a range of 4.6 to 5.3, and more preferably 4.7 to 5.2, throughout the step (b).
The present invention also provides a process for obtaining a series of liquid fractions, which are separated from a culture of A. camphorata over a desired pharmacological activity, such as an anti-tumor activity. Therefore, the third aspect of the present invention is to provide a process for obtaining a pharmacologically active composition from a culture of A. camphorata, comprising:
(a) inoculating a mycelial inoculum of an isolate of A. camphorata into a medium suitable for growth of said isolate;
(b) cultivating the culture resulting from step (a);
(c) removing a major portion of insoluble substances from the culture, whereby a pharmacologically active solution is harvested; and
(d) processing the solution from step (c) so as to obtain a pharmacologically active composition containing fungal molecules having a molecular weight of no more than about 10 kDa.
Preferably, the obtained composition contains fungal molecules having a molecular weight of no more than about 3 kDa, more preferably of no more than about 1 kDa.
The fourth aspect of the present invention is to provide a process for obtaining a pharmacologically active composition from a culture of A. camphorata, comprising:
(a) inoculating a mycelial inoculum of an isolate of A. camphorata into a medium suitable for growth of said isolate;
(b) cultivating the culture resulting from step (a);
(c) removing a major portion of insoluble substances from the culture, whereby an aqueous solution is harvested;
(d) processing the aqueous solution from step (c) so as to obtain a fraction containing fungal molecules having molecular weights of no more than about 1 kDa: and
(e) passing the fraction from step (d) through a water-immiscible phase from which the pharmacologically active composition is obtained.
The water-immiscible phase in the above step (e) is preferably a stationary phase containing an effective amount of an absorbent capable of selectively adsorbing hydrophobic fungal molecules. The stationary phase is eluted so that the pharmacologically active fraction is obtained.
In a preferred embodiment of the invention, the eluate from step (e) is further subjected to reverse-phase partition chromatography, such as on a Lichrosorb(copyright) RP-18 column (Merck), to obtain a number of fractions with pharmacological activity.
The present invention further provides pharmaceutical compositions for the treatment of cancer or tumor diseases, which contains a product obtained from any one of the processes according to the present invention.
The present invention further provides a method for the treatment of cancer or tumor diseases in patients in need of such treatment by prescribing the patients with a composition containing a product obtained from any one of the processes according to the present invention.